Endosomal trafficking of receptors plays fundamental roles in neuronal function and in neurodevelopment, as well as in disease states of the nervous system. The current understanding of endosome organization and function is based primarily on work in yeast and fibroblasts. However, our recent work showed that neurons employ neuronal-specific endocytic and endosomal machinery. There is thus presently a fundamental gap in the understanding of how endosomal function is adapted to cater to the specific physiological needs of neurons. Since the particular organization of the endosome determines postendocytic trafficking of receptors (i.e. signaling, processing, and/or degradation) and thereby specific functional outcomes, the field's ignorance of the neuronal adaptations of the endosomal system constitutes a significant barrier to progress in both basic and disease-oriented fields. Our long-term goals are to uncover the functional contributions of endosomes to neurodevelopment, and to the healthy and diseased brain. The rationale motivating this proposal is that vertebrate neurons express neuronal-specific proteins in their endosomes (in particular NEEP21/Nsg-1 and P19/Nsg-2) that control the endosomal trafficking of crucial neuronal membrane proteins, such as receptors important in development (the axonal adhesion molecule L1/NgCAM), synaptic function (GluA2), and disease (bAPP). Nsg-proteins are members of a gene family of transmembrane proteins found specifically in the neuronal Golgi and in poorly characterized somatodendritic endosomes (Nsg-endosomes). The central concept of this application is that crucial neuronal functions depend on Nsg-endosomes. Our preliminary data suggest the specific hypothesis that Nsg-proteins maintain a specific subset of endocytosed receptors in a distinct non-degradative compartment from where the cargo can recycle to multiple locations to regulate axon growth and synaptic function. We will use innovative approaches including quantitative single vesicle live imaging, super-resolution fluorescence microscopy, and electron microscopy in combination with functional interference approaches in primary neurons and in rodent cortex to address three specific aims: Aim 1) How are Nsg-endosomes formed and how do they relate to other somatodendritic endosomes? With which other proteins does NEEP21/Nsg-1 interact and which rabs regulate Nsg-endosomal organization? Aim 2) How does loss of NEEP21/Nsg-1 and P19/Nsg-2 affect endosomal organization and cargo trafficking? Aim 3) Does loss of NEEP21/Nsg-1 and P19/Nsg-2 affect axon and dendrite development in the cortex? The proposed research is significant because it will discover the contribution of endosomes to neuronal function in health and disease. The new insights gained will not only lead to fundamental advances in understanding the regulation of neuronal membrane traffic, but also raise the possibility of new targets for tailoring translational strateges in the future.